GFP with a 6X His tag for easier protein purification.
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 15 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 15 AA linker as spacer (BBa_K1680003). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
Lactose/IPTG inducible promoter with GFP reporter tagged with AAV-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.
Part was assembled using BrickClip assembly (BBF RFC104) using other biobrick parts as templates. BrickClip assembly is a special case of more general Paperclip assembly method.[1]
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with intermediate 6 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 12 AA linker as spacer (BBa_K1680002). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
sfGFP fused to dCBD with linker in RFC 25
Lactose/IPTG inducible promoter with GFP reporter tagged with DAS-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity. Presence of SspB is crucial for effective protein degradation.
Copper Storage protein 1 (Csp1) is a tetrameric copper storage protein found in the periplasm of Methylosinus trichosporium OB3b. We investigated whether this part could act as a copper chelator when expressed in E. coli. We modified the protein by adding a TAT signal peptide from the E. coli enzyme CueO in place of the native TAT sequence and added a c terminal sfGFP fluorescence protein with a C terminal his tag in an attempt to direct the protein to the periplasm. The Csp1 and sfGFP are separated by a short hydrophilic, flexible linker.
Codon optimised for E. coli. linker added in order to allow the Csp1 and sfGFP to ideally fold separately. Hexahistidine purification tag on c-terminus to maintain TAT sequence.
The source organism for the main protein sequence is Methylosinus trichosporium OB3b. The TAT sequence originate from E. coli multi copper oxidase enzyme CueO. We ordered it as codon optimised DNA from IDT.
This is coding sequence of GFP (yeast optimized codons).
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 12 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 9 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Composed of eGFP-rrnB terminator-T7 terminater, canbe used in further assembly.
Mind the 5'UTR if in a complex construct.
GFP, based on E0040, with overall mutation minimized
Sequence produced by the Vanderbilt iGEM 2015 mutation optimization software
pCopA TAT Csp1 sfGFP with divergent CueR
pCopA TAT Csp1 sfGFP with plasmid constitutively expressed CueR
Variant of GFP that folds robustly even when fused to poorly folded proteins. Waldo and other research groups has reported the engineering of a superfolder GFP (sfGFP) that showed increased resistance to denaturation, improved folding kinetics, and increased resistance to aggregation during refolding (Pedelacq et al., 2006; Andrews et al., 2007; Fisher and DeLisa, 2008). sfGFP has proven to be very useful as a scaffold for improved protein detection and tagging both in vivo and in vitro using self-assembled sfGFP fragments (Cabantous et al., 2005b; Cabantous and Waldo, 2006). Furthermore, sfGFP fusions are more soluble than conventional GFP fusions (Wu et al., 2009).
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Tetracycline/aTc inducible promoter with GFP reporter tagged with LAA-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.
Based on the part E0040, but with an LVA degradation tail attached.
LVA degradation tail and prefix/suffix addition by sequential PCR amplifications with specially designed primers.
Based upon GFP from E0040, with LVA degradation tail attached.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (Freiburg-Standard)
Untagged version of gfp from Repressilator reporter. See the [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0040 untagged version] for source information.
Added Freiburg-restriction-sites for in frame assembly.
[http://partsregistry.org/Part:BBa_I13522 BBa_I13522]
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 6 AA linker as spacer (BBa_K1680000). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
This is the standard GFP protein reporter (E0040) cloned with the prefix/suffix required for standard 25 assembly. It can easily be used to create fusion proteins through this method.
This part contains the AgeI and NgoMIV sites as part of it's prefix/suffix that allows it to be used in standard 25 assembly. It was synthesized through PCR oligo synthesis methods using the following primers: Forward (56.88 degrees) TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCCGTAAAGGAGAAGAACTTTTC Reverse (56.95 degrees) AATACTGCAGCGGCCGCTACTAGTATTAACCGGTCTTGTCGTCATCATCTTTATAAT
This part is similar to the part BBa_E0040 commonly used as a reporter.
The first two nucleotides of the GFP start codon were removed to create this fusion brick.
The GFP fusion brick was derived from the GFP BioBrick, [http://partsregistry.org/Part:BBa_E0040 BBa_E0040].
Tetracycline/aTc inducible promoter with GFP reporter tagged with LVA-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.
Protein coding sequence for a mutant (K145N) of the fluorescent protein Dronpa, codon optimized for Yeast. The mutation leads to photoswitchable monomerisation/multimerisation behaviour (as described by Zhou et al. 2012).
Tetracycline/aTc inducible promoter with GFP reporter tagged with DAS-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.Presence of SspB is crucial for effective protein degradation.
MymT is a small prokaryotic copper metallothein discovered in Mycobacterium tuberculosis. It is believed that the protein may help the bacterium survive copper toxicity, though deleting the gene had no effect on pathogenicity in mice. It can bind up to 7 copper ions but has a preference for 4-6. This version has a C terminal sfGFP with a C terminal hexahistidine tag for purification. The MymT and sfGFP are separated by a small flexible linker. This part has been codon optimised for E. coli.
This protein has a C terminal hexahistidine tag for purification and has been codon optimised for E. coli.
Mycobacterium tuberculosis. Ordered as codon optimised DNA.
pCopA MymT sfGFP with plasmid constitutively expressed CueR
GFP device responsive to environmental stresses
This device takes environmental stresses as input a transcriptional signal (PoPS) and produce as output the fluorescent protein GFP.
This part was assembled using 3A assembly method.
This part is an assembly of promoter uspA (BBa_K1620000) and GFP generator device (BBa_E0840).
Green fluorescent protein (GFP) with N-terminal His-Tag
Jasmin Weber, Michelle Zoeller, Marcus Geissler, Stefan Bohn, Simon Linder, Sebastian Barthel
This is the copper sensitive promoter pCopA (controlled by CueR) in front of an RBS and a sfGFP reporter
Green Fluorescent Protein with LVA tag (GFPLVA)
GFP with LVA-tag can be degrade by speedy degrager and has a short half-life
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (His-tag)
Yeast optimized superfolder GFP C-terminus (215-238)
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the C terminus of CpxR).
We had to test both attachments, N and C terminus of the CpxR.
The arabinose promoter and the sfGFP were obtained from the registry, while the CpxR was obtained from the E coli genome.
Upon recognition of a small molecule (e.g. nutrients or toxins), chemoreceptors transduce a signal through a set of Che proteins. This group of methylesterases and phosphatases regulates the rotation of the flagella. At the end of the chain of Che proteins is CheY. In phosphorylated state, CheY binds the flagella and causes the cells to tumble. The phosphatase CheZ dephosphorylates CheY, inducing dissociation of CheY from the flagella enabling the cells to swim. Cells lacking the CheZ protein are unable to swim and will tumble excessively and incessantly.
1 bp in BBa_K629003 was adapted to create an original restriciton site in the plasmid. CheZ was fused to GFP (BBa_K082003) and its LVA tag was codon-optimized to avoid introducing artificial DNA repetitions.
DNA sequences originate from the E. Coli genome and previous biobrick sequences.
we REMOVED the extra base from part BBa_I715020(easier to fuse with other proteins) when designing PCR primers to make the protein fusion easier.
This part is gained from part BBa_I715020.
Naringenin sensor (FdeR) with GFP as reporter
Sascha Hein, Niklas Hummel, Sebastian Barthel, Thomas Dohmen
We modified the naringenin biosensor from Siedler et al. by elimination of one EcoRI restriction site and by changing the native ribosomal binding site into the strong RBS Bba_B0034. Based on this reporter protein and 3 different fluorescence proteins, we designed 3 new biosensors for in vivo detection and determination of naringenin. The natural source of FdeR is Herbaspirillum seropedicae. You can use our reporter for measuring naringenin concentrations in your samples. Depending on which fluorophor you want to detect, you can use one of three biosensors: GFP fluorescence K1497020 mKate (DsRed variant) fluorescence K1497021 CFP fluorescence K1497022 You can create your own naringenin sensor or your own naringenin dependent gene expression device as well. For these reasons use FdeR Biobrick K1497019 and clone your parts of interest (without RBS!) behind the device.
The structure of toehold switch is similar to hairpin, except it has a loop at the top as ???toehold???. Toehold switch functions as riboregulator through linear-linear interaction between RNAs. When target RNA appears, it will bind one of the toehold switch stems and open the loop, exposing the RBS. Toehold switch systems are composed of two RNA strands referred to as the switch and trigger. The switch RNA contains the coding sequence of the gene being regulated. Upstream of this coding sequence is a hairpin-based processing module containing both a strong RBS and a start codon that is followed by a common 21 nt linker sequence coding for low-molecular-weight amino acids added to the N terminus of the gene of interest. A single-stranded toehold sequence at the 50 end of the hairpin module provides the initial binding site for the trigger RNA strand. This trigger molecule contains an extended single-stranded region that completes a branch migration process with the hairpin to expose the RBS and start codon, thereby initiating translation of the gene of interest. The trigger RNA of our toehold is miRNA-144.When adding it to the system, GFP can be expressed.
Toehold can be open by trigger RNA, thus letting GFP express.
GFP (mut3b) with LVA-ssrA degradation tag
GFP (mut3b) (see part BBa_E0040) with added LVA-ssrA degradation tag. The tag increases GFP turn-over rate, thus providing better temporal resolution of green fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALVA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LVA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations, protein stability, Km of binding to the protease, temperature [3]. References [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to GFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LVA in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
GFP comes from part E1010, the tag sequence was obtained from paper by Andersen et al., (1998).[2]
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the N terminus of CpxR).
From Pectinidae sp. It is a CDS of green fluorescent protein Dronpa.
Pectinidae sp. sequence found in the following article Lummer, M., Humpert, F., Steuwe, C., Caesar, K., Sch??ttpelz, M., Sauer, M. and Staiger, D. (2011), Reversible Photoswitchable DRONPA-s Monitors Nucleocytoplasmic Transport of an RNA-Binding Protein in Transgenic Plants. Traffic, 12: 693???702. doi: 10.1111/j.1600-0854.2011.01180.x
sfGFP in Freiburg format (RFC 25) to allow for easy use in fusion proteins.
It generates permanently and a low rate GFP reporter. It is usefull for cell marker and promoter strength tests.
This part is assembled from parts BBa_J23101 and GFP generator (BBa_E0840).
GoldenGate compatible Green Flourescent Protein (GFP)
A variation of the part BBa_E0040 that has been made compatible for Golden Gate by removal of internal BsaI and BpiI recognition sequences. Fluoresces under UV light when the gene is expressed.
Domestication for GoldenGate by removal of internal BsaI and BpiI recognition sequences.
dnMyD88 is a dominant negative form of protein MyD88 (an important protein in signaling cascade through TLR4 receptor). dnMyD88 is linked to GFP (acts as a reporter) through six aminoacid long protein linker.
dnMyD88-linker-GFP was made by using PCR overlap extension technique. This part is cloned in BioBrick vector pSB1AK3. Original sequence has PstI restriction site so we have to introduce a mutation without affect on amino acid sequence.
Tetracycline/aTc inducible promoter with GFP reporter tagged with NYADAS-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.
Pu promoter and RBS from GLuc coding device with Pu (BBa_K2023003) and GFP reporter for RHS of library test constructs (BBa_I13401)
A CDS of Dronpa protein with a mutation in the betha-strand 7 in Lys145 for Asn.
EGFP (BBa_1491002) with 6X-His tag for easier protein purification. Original sequence is codon optimized by IDT Codon Optimized Tool for E.coli.
Codon optimized for E.coli and His tag allows for easier protein purification.
This is the coding sequence of a yeast optimized GFP with BD ends for AarI cloning.
As an AD part, there is no ATG, and the stop codon is included. For more information on AarI cloning, please refer to the UCSF 2008 igem page.
This part is a reporter sfGFP(Sp) originally optimized for Streptococcus pneumoniae.
sfGFP originally optimized for streptococcus pneumonia because it could be shown that the signal in Bacillus is even brighter than the one from sfGFP(Bs).
Yeast optimized sfGFP N-terminus (1-214)
Tetracycline/aTc inducible promoter with GFP reporter tagged with AAV-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.
Part is composed of TIR domain of Traf6 and GFP linked with 6 aminoacids long linker. GFP serves as reporter. For usage in eukaryotes.
This part is the Amino Half of GFP with termination codon
Because of the need of our project, we added a termination codon when designing the PCR.
This part is gained from part BBa_I715019. we added the termination codon when designing the PCR.
DNA sequences originate from the E. Coli genome and previous biobrick sequences. The ribosome binding site was chosen from the Anderson RBS family.
P-Free JuniperGFP (Green Fluorescent Protein)
This part was created by DNA 2.0 as part of their IP-Free series of fluorescent and chromogenic proteins. It is available to use under the BioBrick Public Agreement. Excitation 508nm, Emission 521nm Molecular Weight: 26.6kDA Length: 237aa
This sequence is fully synthetic, (non-Aequorea) and is codon optimized for expression in E. coli.
This synthetic part was created through a gene engineering and synthesis project at DNA 2.0.
Lactose/IPTG inducible promoter with GFP reporter tagged with NYADAS-ssrA degradation tag followed by terminator. The tagged GFP is actively degraded within cell, thus provides better temporal resolution of green fluorescence and promoter activity.